The overall objective of this project is to develop a novel chemical class of broad-spectrum therapeutic agents, the bis-(indole) lead series, for use against Gram-negative nonfermenters. The limited number and efficacy of current therapies, the scarcity of novel broad-spectrum antibiotics in the drug development pipeline and the ease with which these bacteria develop drug resistance, argue for the urgent development of new more potent, broad-acting antibacterial agents capable of combating these pathogens. Compounds in this new chemical series exhibit broad-spectrum antibacterial activity, displaying potent (MIC's of 0.2-3 ?g/ml) inhibition of the growth of Gram-negative bacterial species, including such Gram-negative nonfermenters as Acinetobacter baumannii, Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia. These compounds are also active against fastidious Gram- negative bacilli and a large number of strains from the Enterobacteriaceae family as well as a variety of Gram-positive bacterial species. They act by a rapid bactericidal mechanism, exhibiting 3-log's of killing in 1-4 hours at concentrations near the MIC values. The precise mechanism of action is unknown;however, the lead series has been shown to inhibit DNA replication in permeabilized bacterial cells and to inhibit purified bacterial replicative DNA helicase. We have not been able to isolate mutants resistant to the primary lead scaffold MBX 1066 or its analog MBX 1162. The lead series is effective in murine models of Gram-negative infections (i.e., Yersinia pestis, Burkholderia pseudomallei;ED50 <10 mg/kg). In addition, the compounds are well-tolerated in mice (MTD =400 mg/kg). Finally, these compounds are relatively easy and inexpensive to synthesize, with a very favorable cost of goods. These overall characteristics advocate for the rapid development of these compounds as broad spectrum antibacterial agents for use against difficult-to-treat Gram-negative bacterial pathogens. We will chemically optimize the bis-(indole) lead series in a rational drug discovery effort focused initially on improving broad-spectrum potency while minimizing mammalian cytotoxicity. We will demonstrate efficacy in vivo in a murine model of A. baumannii-induced pneumonia. In Phase II of this project, we will expand upon the in vivo characterization with at least two rat infection models (pneumonia and skin pouch) and then conduct IND-enabling (GLP) pharmacokinetic, toxicology and safety pharmacology studies in two species suitable for IND submission. PUBLIC HEALTH RELEVANCE: Infectious disease caused by the Gram-negative nonfermenters, such as Acinetobacter baumannii, are difficult to treat due to the frequency of multi-drug resistance in these organisms. They are increasingly found in hospital and community-acquired infections as well as those infections obtained in the battlefields of Iraq and Afghanistan. This proposal describes a rational approach for targeting these bacterial agents with the bis-(imidazolinylindole) series of compounds, a series of potent antibacterials, discovered by a group at USAMRIID and Microbiotix, by creating more potent compounds and testing them for in vitro and in vivo activity.